Characterization of Arnebia euchroma PGT homologs involved in the biosynthesis of shikonin.

Shikonin is a red naphthoquinone natural product from plants with high economical and medical values. The para-hydroxybenzoic acid geranyltransferase (PGT) catalyzes the key regulatory step of shikonin biosynthesis. PGTs from Lithospermum erythrorhizon have been well-characterized and used in industrial shikonin production. However, its perennial medicinal plant Arnebia euchroma accumulates much more pigment and the underlying mechanism remains obscure. Here, we discovered and characterized the different isoforms of AePGTs. Phylogenetic study and structure modeling suggested that the N-terminal of AePGT6 contributed to its highest activity among 7 AePGTs. Indeed, AePGT2 and AePGT3 fused with 60 amino acids from the N-terminal of AePGT6 showed even higher activity than AePGT6, while native AePGT2 and AePGT3 don't have catalytic activity. Our result not only provided a mechanistic explanation of high shikonin contents in Arnebia euchroma but also engineered a best-performing PGT to achieve the highest-to-date production of 3-geranyl-4-hydroxybenzoate acid, an intermedium of shikonin.

[Dynamic control of ERG20 expression to improve production of monoterpenes by engineering Saccharomyces cerevisiae].

Monoterpenes are widely used in cosmetics, food, medicine, agriculture and other fields. With the development of synthetic biology, it is considered as a potential way to create microbial cell factories to produce monoterpenes. Engineering Saccharomyces cerevisiae to produce monoterpenes has been a research hotspot in synthetic biology. In S. cerevisiae, the production of geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) is catalyzed by a bifunctional enzyme farnesyl pyrophosphate synthetase(encoded by ERG20 gene) which is inclined to synthesize FPP essential for yeast growth. Therefore, reasonable control of FPP synthesis is the basis for efficient monoterpene synthesis in yeast cell factories. In order to achieve dynamic control from GPP to FPP biosynthesis in S. cerevisiae, we obtained a novel chassis strain HP001-pERG1-ERG20 by replacing the ERG20 promoter of the chassis strain HP001 with the promoter of cyclosqualene cyclase(ERG1) gene. Further, we reconstructed the metabolic pathway by using GPP and neryl diphosphate(NPP), cis-GPP as substrates in HP001-pERG1-ERG20. The yield of GPP-derived linalool increased by 42.5% to 7.6 mg·L~(-1), and that of NPP-derived nerol increased by 1 436.4% to 8.3 mg·L~(-1). This study provides a basis for the production of monoterpenes by microbial fermentation.

[Cloning of transcription factor PcFBA-1 in Pogostemon cabin and its interaction with FPPS promoter].

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

Comparative Studies on Bioactive Compounds, Ganoderic Acid Biosynthesis, and Antioxidant Activity of Pileus and Stipes of Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes) Fruiting Body at Different Growth Stages.

Total phenolics, flavonoids, and polysaccharides, and individual ganoderic acid (GA) contents, antioxidant capacity, and transcription levels of key enzyme genes involved in GA biosynthesis in pileus and stipes of Ganoderma lucidum fruiting body at different growth stages were investigated in this study. Results showed that the highest total phenolics and total flavonoids contents were determined in stipes at spore maturity stage, resulting in high antioxidant activity, while the highest total polysaccharide content was found in pileus at the same stage. The pileus contained more GA than the stipes, and higher contents of ganoderic acid A and D were found at fruiting body mature stage while that of ganoderic acid B, C2, and G were found at bud elongation stage. Results from quantitative real-time PCR indicated that higher gene transcription levels of hydroxyl methylglutaryl-CoA reductase (hmgr), farnesyl pyrophosphate synthase (fps), squalene synthase (sqs), and oxidosqualene cyclase (osc) were found in pileus at bud elongation stage. Our findings will be helpful for understanding the biosynthesis of bioactive components and determining the harvest time for the desired G. lucidum fruiting bodies.

NMR for screening and a biochemical assay: Identification of new FPPS inhibitors exerting anticancer activity.

Farnesyl pyrophosphate synthase (FPPS) is a crucial enzyme for the synthesis of isoprenoids and the key target of nitrogen-containing bisphosphonates (N-BPs). N-BPs are potent and selective FPPS inhibitors that are used in the treatment of bone-related diseases, but have poor pharmacokinetic properties. Given the key role played by FPPS in many cancer-related pathways and the pharmacokinetic limits of N-BPs, hundreds of molecules have been screened to identify new FPPS inhibitors characterized by improved drug-like properties that are useful for broader therapeutic applications in solid, non-skeletal tumours. We have previously shown that N6-isopentenyladenosine (i6A) and its related compound N6-benzyladenosine (2) exert anti-glioma activity by interfering with the mevalonate pathway and inhibiting FPPS. Here, we report the design and synthesis of a panel of N6-benzyladenosine derivatives (compounds 2a-m) incorporating different chemical moieties on the benzyl ring. Compounds 2a-m show in vitro antiproliferative activity in U87MG glioma cells and, analogous to the bisphosphonate FPPS inhibitors, exhibit immunogenic properties in ex vivo γδ T cells from stimulated peripheral blood mononuclear cells (PBMCs). Using saturation transfer difference (STD) and quantitative 1H nuclear magnetic resonance (NMR) experiments, we found that 2f, the N6-benzyladenosine analogue that includes a tertbutyl moiety in the para position of the benzyl ring, is endowed with increased FPPS binding and inhibition compared to the parent compounds i6A and 2. N6-benzyladenosine derivatives, characterized by structural features that are significantly different from those of N-BPs, have been confirmed to be promising chemical scaffolds for the development of non N-BP FPPS inhibitors, exerting combined cytotoxic and immunostimulatory activities.

[Correlation study between accumulation of triterpenoids and expression of relative genes in Alisma orientale].

To research the correlation between accumulation of triterpenoids and expression of key enzymes genes in triterpenoid biosynthesis of Alisma orientale,the study utilized UPLC-MS/MS method to detect eight triterpenoids content in the tuber of A. orientale from different growth stages,including alisol A,alisol A 24 acetate,alisol B,alisol B 23 acetate,alisol C 23 acetate,alisol F,alisol F 24 acetate and alisol G,and then the Real time quantitative PCR was used to analyze the expression of key enzymes genes HMGR and FPPS in triterpenoid biosynthesis. Correlation analysis showed that there was a significant positive relation between the total growth of these eight triterpenoids and the average relative expression of HMGR and FPPS(HMGR: r = 0. 998,P<0. 01; FPPS: r = 0. 957,P<0. 05),respectively. Therefore,the study preliminarily determined that HMGR and FPPS genes could regulate the biosynthesis of triterpenoids in A. orientale,which laid a foundation for further research on the biosynthesis and regulation mechanism of triterpenoids in A. orientale.

Effect of nanoparticle treatment on expression of a key gene involved in thymoquinone biosynthetic pathway in Nigella sativa L.

Thymoquinone is the most important secondary metabolite in black Cumin, which has several pharmaceutical applications. In this study, effect of TiO2 and SiO2 nanoparticles as new elicitors, on expression of Geranyl diphosphate synthase gene (GPPS gene), as a key gene involved in thymoquione biosynthesis pathway was investigated in two Iranian accessions. Plants were treatment in the early flowering stage and after 24 h of 50 and 100 mg/L of each nanoparticle, separately. After RNA extraction, GPPS gene expression was analysed by qRT-PCR method. The results showed that the TiO2 and SiO2 nanoparticles, generally stimulates the GPPS expression. The TiO2 nanoparticles were more effective than SiO2 for the induction of GPPS expression. Also, 100 mg/L treatment of nanoparticles raised gene expression more than 50 mg/L concentration. It can be concluded these nanoparticles can be used as robust elicitors to enhance the production of Thymoquinone in black cumin through up-regulation of related metabolic pathway genes.

A farnesyl pyrophosphate synthase gene expressed in pollen functions in S-RNase-independent unilateral incompatibility.

Multiple independent and overlapping pollen rejection pathways contribute to unilateral interspecific incompatibility (UI). In crosses between tomato species, pollen rejection usually occurs when the female parent is self-incompatible (SI) and the male parent self-compatible (SC) (the 'SI × SC rule'). Additional, as yet unknown, UI mechanisms are independent of self-incompatibility and contribute to UI between SC species or populations. We identified a major quantitative trait locus on chromosome 10 (ui10.1) which affects pollen-side UI responses in crosses between cultivated tomato, Solanum lycopersicum, and Solanum pennelliiLA0716, both of which are SC and lack S-RNase, the pistil determinant of S-specificity in Solanaceae. Here we show that ui10.1 is a farnesyl pyrophosphate synthase gene (FPS2) expressed in pollen. Expression is about 18-fold higher in pollen of S. pennellii than in S. lycopersicum. Pollen with the hypomorphic S. lycopersicum allele is selectively eliminated on pistils of the F1 hybrid, leading to transmission ratio distortion in the F2 progeny. CRISPR/Cas9-generated knockout mutants (fps2) in S. pennelliiLA0716 are self-sterile due to pollen rejection, but mutant pollen is fully functional on pistils of S. lycopersicum. F2 progeny of S. lycopersicum × S. pennellii (fps2) show reversed transmission ratio distortion due to selective elimination of pollen bearing the knockout allele. Overexpression of FPS2 in S. lycopersicum pollen rescues the pollen elimination phenotype. FPS2-based pollen selectivity does not involve S-RNase and has not been previously linked to UI. Our results point to an entirely new mechanism of interspecific pollen rejection in plants.

Structure-Function Studies of Artemisia tridentata Farnesyl Diphosphate Synthase and Chrysanthemyl Diphosphate Synthase by Site-Directed Mutagenesis and Morphogenesis.

The amino acid sequences of farnesyl diphosphate synthase (FPPase) and chrysanthemyl diphosphate synthase (CPPase) from Artemisia tridentata ssp. Spiciformis, minus their chloroplast targeting regions, are 71% identical and 90% similar. FPPase efficiently and selectively synthesizes the "regular" sesquiterpenoid farnesyl diphosphate (FPP) by coupling isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) and then to geranyl diphosphate (GPP). In contrast, CPPase is an inefficient promiscuous enzyme, which synthesizes the "irregular" monoterpenes chrysanthemyl diphosphate (CPP), lavandulyl diphosphate (LPP), and trace quantities of maconelliyl diphosphate (MPP) from two molecules of DMAPP, and couples IPP to DMAPP to give GPP. A. tridentata FPPase and CPPase belong to the chain elongation protein family (PF00348), a subgroup of the terpenoid synthase superfamily (CL0613) whose members have a characteristic α terpene synthase α-helical fold. The active sites of A. tridentata FPPase and CPPase are located within a six-helix bundle containing amino acids 53 to 241. The two enzymes were metamorphosed into one another by sequentially replacing the loops and helices of the six-helix bundle from enzyme with those from the other. Chain elongation was the dominant activity during the N-terminal to C-terminal metamorphosis of FPPase to CPPase, with product selectivity gradually switching from FPP to GPP, until replacement of the final α-helix, whereupon cyclopropanation and branching activity competed with chain elongation. During the corresponding metamorphosis of CPPase to FPPase, cyclopropanation and branching activities were lost upon replacement of the first helix in the six-helix bundle. Mutations of active site residues in CPPase to the corresponding amino acids in FPPase enhanced chain-elongation activity, while similar mutations in the active site of FPPase failed to significantly promote formation of significant amounts of irregular monoterpenes. Our results indicate that CPPase, a promiscuous enzyme, is more plastic toward acquiring new activities, whereas FPPase is more resistant. Mutations of residues outside of the α terpene synthase fold are important for acquisition of FPPase activity for synthesis of CPP, LPP, and MPP.

[Cloning and protein expression analysis of geranyl diphosphate synthase genes in Tripterygium wilfordii].

Based on the transcriptome data, the study cloned full-length cDNA of TwGPPS1 and TwGPPS2 genes from Tripterygium wilfordii suspension cells and then analyzed the bioinformation of the sequence and protein expression. The cloned TwGPPS1 has a 1 278 bp open reading frame (ORF) encoding a polypeptide of 425 amino acids. The deduced isoelectric point (pI) was 6.68, a calculated molecular weight was about 47.189 kDa. The full-length cDNA of the TwGPPS2 contains a 1 269 bp open reading frame (ORF) encoding a polypeptide of 422 amino acids. The deduced isoelectric point (pI) was 6.71, a calculated molecular weight was about 46.774 kDa.The entire reading frame of TwGPPS1,2 was cloned into the pET-32a(+) vector and expressed in E. coli BL21 (DE3) cells to obtain the TwGPPS protein, which laid a basis for further study on the regulation of terpenoid secondary metabolism and biological synthesis.

Production of 3-geranyl-4-hydroxybenzoate acid in yeast, an important intermediate of shikonin biosynthesis pathway.

Shikonin and its derivatives are the main active components in the medicinal plant Arnebia euchroma and possess extensive pharmaceutical properties. In this study, we developed an optimized yeast system to obtain high-level production of 3-geranyl-4-hydroxybenzoate acid (GBA), an important intermediate involved in shikonin biosynthesis pathway. For host selection, recombinant expression of p-hydroxybenzoate:geranyltransferase (PGT) derived from A. euchroma was performed in Saccharomyces cerevisiae WAT11U strain and high yield monoterpene strain. In shake flask culture with 1 mM p-hydroxybenzoate acid (PHBA), they could yield GBA at 0.1567 and 20.8624 mg L-1, respectively. Additionally, AePGT6 showed higher enzymatic activity than its homologs. Moreover, by combining improvement in the homologous mevalonate pathway with reconstruction in the heterologous shikimic pathway, a de novo GBA synthesis pathway was constructed in StHP6tHC with co-overexpressed SctHMG1, optimized EcUbiC and AePGT6. A high titer of 179.29 mg L-1 GBA was achieved in StHP6tHC under shake flask fermentation with 1 mM PHBA. These results suggest that yeast could be engineered systematically to enable an efficient monoterpene-quinone or naphthoquinone production.

Synergistic Activity between Statins and Bisphosphonates against Acute Experimental Toxoplasmosis.

Bisphosphonates are widely used for the treatment of bone disorders. These drugs also inhibit the growth of a variety of protozoan parasites, such as Toxoplasma gondii, the etiologic agent of toxoplasmosis. The target of the most potent bisphosphonates is the isoprenoid biosynthesis pathway enzyme farnesyl diphosphate synthase (FPPS). Based on our previous work on the inhibitory effect of sulfur-containing linear bisphosphonates against T. gondii, we investigated the potential synergistic interaction between one of these derivatives, 1-[(n-heptylthio)ethyl]-1,1-bisphosphonate (C7S), and statins, which are potent inhibitors of the host 3-hydroxy-3-methyl glutaryl-coenzyme A reductase (3-HMG-CoA reductase). C7S showed high activity against the T. gondii bifunctional farnesyl diphosphate (FPP)/geranylgeranyl diphosphate (GGPP) synthase (TgFPPS), which catalyzes the formation of FPP and GGPP (50% inhibitory concentration [IC50] = 31 ± 0.01 nM [mean ± standard deviation]), and modest effect against the human FPPS (IC50 = 1.3 ± 0.5 µM). We tested combinations of C7S with statins against the in vitro replication of T. gondii We also treated mice infected with a lethal dose of T. gondii with similar combinations. We found strong synergistic activities when using low doses of C7S, which were stronger in vivo than when tested in vitro We also investigated the synergism of several commercially available bisphosphonates with statins both in vitro and in vivo Our results provide evidence that it is possible to develop drug combinations that act synergistically by inhibiting host and parasite enzymes in vitro and in vivo.

Metabolic engineering of Saccharomyces cerevisiae for production of germacrene A, a precursor of beta-elemene.

Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Engineering Escherichia coli to convert acetic acid to ß-caryophyllene.

BACKGROUND: Under aerobic conditions, acetic acid is the major byproduct produced by E. coli during the fermentation. And acetic acid is detrimental to cell growth as it destroys transmembrane pH gradients. Hence, how to reduce the production of acetic acid and how to utilize it as a feedstock are of intriguing interest. In this study, we provided an evidence to produce ß-caryophyllene by the engineered E. coli using acetic acid as the only carbon source. RESULTS: Firstly, to construct the robust acetate-utilizing strain, acetyl-CoA synthases from three different sources were introduced and screened in the E. coli. Secondly, to establish the engineered strains converting acetic acid to ß-caryophyllene, acetyl-CoA synthase (ACS), ß-caryophyllene synthase (QHS1) and geranyl diphosphate synthase (GPPS2) were co-expressed in the E. coli cells. Thirdly, to further enhance ß-caryophyllene production from acetic acid, the heterologous MVA pathway was introduced into the cells. What's more, acetoacetyl-CoA synthase (AACS) was also expressed in the cells to increase the precursor acetoacetyl-CoA and accordingly resulted in the increase of ß-caryophyllene. The final genetically modified strain, YJM67, could accumulate the production of biomass and ß-caryophyllene up to 12.6 and 1.05 g/L during 72 h, respectively, with a specific productivity of 1.15 mg h(-1) g(-1) dry cells, and the conversion efficiency of acetic acid to ß-caryophyllene (gram to gram) reached 2.1%. The yield of ß-caryophyllene on acetic acid of this strain also reached approximately 5.6% of the theoretical yield. CONCLUSIONS: In the present study, a novel biosynthetic pathway for ß-caryophyllene has been investigated by means of conversion of acetic acid to ß-caryophyllene using an engineered Escherichia coli. This was the first successful attempt in ß-caryophyllene production by E. coli using acetic acid as the only carbon source. Therefore, we have provided a new metabolic engineering tool for ß-caryophyllene synthesis.

Monoterpene biosynthesis potential of plant subcellular compartments.

Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana benthamiana indicated local GDP availability for each compartment but resulted in different product levels. A GDP synthase from Picea abies (PaGDPS1) was shown to boost GDP production. PaGDPS1 was also targeted to plastids, cytosol or mitochondria and PaGDPS1 and GES were coexpressed in all possible combinations. Geraniol and geraniol-derived products were analyzed by GC-MS and LC-MS, respectively. GES product levels were highest for plastid-targeted GES, followed by mitochondrial- and then cytosolic-targeted GES. For each compartment local boosting of GDP biosynthesis increased GES product levels. GDP exchange between compartments is not equal: while no GDP is exchanged from the cytosol to the plastids, 100% of GDP in mitochondria can be exchanged to plastids, while only 7% of GDP from plastids is available for mitochondria. This suggests a direct exchange mechanism for GDP between plastids and mitochondria. Cytosolic PaGDPS1 competes with plastidial GES activity, suggesting an effective drain of isopentenyl diphosphate from the plastids to the cytosol.

[Transcriptome-based gene mining and bioinformatics analysis of p-hydroxybenzoate geranyltransferase genes in Arnebia euchroma].

The p-hydroxybenzoate geranyltransferases(PGT) play an important role in the biosynthesis pathways of shikonin derivatives. Six PGTs were obtained from transcriptome datebase of Arnebia euchroma by using bioinformatics methods and the proteins＇physiochemical properties they encoded were predicted. The result of protein domain prediction showed all of the six protein sequences contained the conserved domain of Ubia prenyltransferase family and possessed the motif NDxxDxxxD for prenyldiphosphate binding and a GX(K/Y)STAL sequence for putative aromatic ring binding. The phylogenetic tree showed that PGT and p-hydroxybenzoate polyprenyltransferase(PPT) belonged to two different clades. The results of gene expression analyses showed that the expression levels in the red shikonin-proficient line and the overground part of A. euchroma that could produce shikonin derivatives was much higher than the white shikonin-deficient line and the underground part, which suggested a positive correlation between the expression levels of PGT genes and shikonin production. This study aims to lay a foudation for further understanding of the function and enzymatic properties of PGT and provide a basis for the biosynthesis pathways and metabolic regulation of shikonin derivatives.

Enhanced Production of δ-Guaiene, a Bicyclic Sesquiterpene Accumulated in Agarwood, by Coexpression of δ-Guaiene Synthase and Farnesyl Diphosphate Synthase Genes in Escherichia coli.

Two genes involved in δ-guaiene biosynthesis in Aquilaria microcarpa, δ-guaiene synthase (GS) and famesyl diphosphate synthase (FPS), were overexpressed in Escherichia coli cells. Immunoblot analysis revealed that the concentration of GS-translated protein was rather low in the cells transformed by solely GS while appreciable accumulation of the recombinant protein was observed when GS was coexpressed with FPS. GS-transformed cells liberated only a trace amount of δ-guaiene (0.004 µg/mL culture), however, the concentration of the compound elevated to 0.08 pg/mL culture in the cells transformed by GS plus FPS. δ-Guaiene biosynthesis was markedly activated when E. coli cells coexpressing GS and FPS were incubated in enriched Terrific broth, and the content of the compound increased to approximately 0.6 µg/mL culture. These results suggest that coexpression of FPS and GS in E. coli is required for efficient δ- guaiene production in the bacterial cells, and the sesquiterpene-producing activity of the transformant is appreciably enhanced in the nutrients-enriched medium.

Functional Characterization of Novel Sesquiterpene Synthases from Indian Sandalwood, Santalum album.

Indian Sandalwood, Santalum album L. is highly valued for its fragrant heartwood oil and is dominated by a blend of sesquiterpenes. Sesquiterpenes are formed through cyclization of farnesyl diphosphate (FPP), catalyzed by metal dependent terpene cyclases. This report describes the cloning and functional characterization of five genes, which encode two sesquisabinene synthases (SaSQS1, SaSQS2), bisabolene synthase (SaBS), santalene synthase (SaSS) and farnesyl diphosphate synthase (SaFDS) using the transcriptome sequencing of S. album. Using Illumina next generation sequencing, 33.32 million high quality raw reads were generated, which were assembled into 84,094 unigenes with an average length of 494.17 bp. Based on the transcriptome sequencing, five sesquiterpene synthases SaFDS, SaSQS1, SaSQS2, SaBS and SaSS involved in the biosynthesis of FPP, sesquisabinene, ß-bisabolene and santalenes, respectively, were cloned and functionally characterized. Novel sesquiterpene synthases (SaSQS1 and SaSQS2) were characterized as isoforms of sesquisabinene synthase with varying kinetic parameters and expression levels. Furthermore, the feasibility of microbial production of sesquisabinene from both the unigenes, SaSQS1 and SaSQS2 in non-optimized bacterial cell for the preparative scale production of sesquisabinene has been demonstrated. These results may pave the way for in vivo production of sandalwood sesquiterpenes in genetically tractable heterologous systems.

Cloning and characterization of a farnesyl pyrophosphate synthase from Matricaria recutita L. and its upregulation by methyl jasmonate.

Matricaria recutita (L.), commonly known as chamomile, is one of the most valuable medicinal plants because it synthesizes a large number of pharmacologically active secondary metabolites known as α-bisabolol and chamazulene. Although the plant has been well characterized in terms of chemical constituents of essential oil as well as pharmacological properties, little is known about the genes responsible for biosynthesis of these compounds. In this study, we report a new full-length cDNA encoding farnesyl diphosphate synthase (FPS), a key enzyme in the pathway of biosynthesis of isoprenoids, from M. recutita. The cDNA of MrFPS comprises 1032 bp and encodes 343 amino acid residues with a calculated molecular mass of 39.4 kDa. The amino acid sequence homology and phylogenetic analysis indicated that MrFPS belongs to the plant FPS super-family and is closely related to FPS from the Asteraceae family. Expression of the MrFPS gene in Escherichia coli yielded FPS activity. Using real-time quantitative PCR, the expression pattern of the MrFPS gene was analyzed in different tissues of M. recutita as well as in response to methyl jasmonate. The expression analysis demonstrated that MrFPS expression varies in different tissues (with maximal expression in flowers and stems) and was significantly elevated in response to methyl jasmonate. This study will certainly enhance our understanding of the role of MrFPS in the biosynthesis and regulation of valuable secondary metabolites in M. recutita at a molecular level.